Preparation of cell nuclei is desirable for a number of subsequent applications. These include studies relating to the mechanisms of transcription in the cell. The nuclei are used to contain trans-acting factors and necessary enzymes and cofactors to allow transcription to occur in in vitro reactions. Soluble extracts from nuclei preparations are useful for both trans-and cis-acting element study as well as being useful for purification and physical characterisation of the factors.
The first report of the use of nuclear extracts was reported in 1983 (Dignam et al N.A.R. 11: 1475-1489) and these have now become standard practice. These methods take 3-4 hours to prepare the nuclei however, involving two spins in an ultra centrifuge using density cushions. The clean intact nuclei are then made into extracts after lysis for their use in experiments such as G-free cassette, (Sawadogo and Roeder P.N.A.S. 82 4394-4398, 1985), primer extension, RNAse mapping, S1 nuclease etc. These traditional methods of nuclei isolation are laborious, slow and limit the number of samples which can be handled at a time. They also compromise the quality of the final lysate in that the nuclei are in the process for long periods during which time many useful factors can leach out or be denatured.
Generally the generation of nuclei is performed by selective lysis of cell cytoplasm by osmotic shock, mechanical shearing and differential centrifugation so the nuclei are the only items to sediment. These processes are very difficult to implement especially in complex biological samples and proscribes against the processing of large numbers of samples and automation. Consequently nuclei preparations are only done when there is no other alternative method although reports in the literature have stated that the quality of DNA derived from purified nuclei is superior to DNA extracted by other means. (A Laboratory Guide for In Vivo Studies of DNA Methylation and Protein/DNA Interactions: Biomethods vol 3 ed. by H. P. Saluz, J. P. Jost 1990 pub. by Birkhauser Verlag).
For the vast majority of procedures in both research and diagnostic molecular biology extracted nucleic acids are required as the first step. For example, relatively pure samples of genomic DNA are required to perform tests for genetic diseases and for recombinant DNA technology the DNA to be cloned must be purified. In the detection of infectious disease organisms such as viruses, access to the cell DNA is necessary where many of these microbes lie hidden.
To avoid the meticulous and lengthy procedures of nuclei preparation, extraction of DNA is commonly done by complete initial release of the DNA by disruption of cytoplasm and nucleus as the first step. This can be done by freeze-thawing, ultrasound, shearing, enzymes, chelating agents or surfactants. DNA is not found as free molecules in a cell nucleus but exists as a complex association of DNA, RNA and proteins. Most DNA extraction techniques use degrading enzymes for major protein and RNA removal followed by repeated solvent extraction with phenol and alcohols to remove residual contaminating proteins and other macromolecules.
These processes are labour intensive, require the use of hazardous, volatile solvents and because of the number of manipulation steps can result in the relatively fragile genomic DNA being broken into small pieces. For many procedures large fragments are necessary (see below). Standard nucleic extraction procedures are mentioned in reference Sambrook et al Molecular Cloning A Laboratory Manual 2nd edition 9.14 (New York: Cold Spring Harbor Laboratory 1989), These involve lysis of cells, enzyme digestions, repeated phenol/chloroform extractions and dialysis, the whole procedure taking many hours. There has been considerable work on developing improved extraction procedures which avoid the use of organic solvents. These include use of chaotropes such as sodium chloride, sodium perchlorate, lithium chloride. [Grimberg J. et al N.A.R. 17, 8390 (1989), Buttone G. J. and Darlington G. J. Clin. Chem. 31, 164-165 (1989), Johns, M. B. and Paulus-Thomas J. E. Anal. Biochem. 180, 276-278 (1989), Miller S. A. N.A.R. 16, 1215 (1988)]
Patents EP-A-0 145 356 and EP-A-0 240 191 and EP-A-0 245 945 describe extraction methods all of which involve alcohol extraction. DNA extraction from blood samples is particularly troublesome as an extra step of separating out the white cells from the vast excess of red cells and serum proteins is usually necessary. This is done by centrifugation either to isolate the buffy coat fraction or to pellet the nuclei after cellular lysis.[Grimberg J. et al N.A.R. 17, 8390 (1989)]
Recent developments for amplification and detection of nucleic acids using polymerase chain reaction and other amplification methods require purified or extracted DNA to a different specification. Patent numbers U.S. Pat. No. 4,683,195 and U.S. Pat. No. 4,683,202 describe amplification and detection procedures for nucleic acids found in various biological specimens using a polymerase. Although DNA for amplification often does not need such thorough purification as for other applications there are other important requirements for blood DNA samples such as the efficient removal of haemoglobin. This and other inhibitory substances in blood must be removed to avoid inhibition of the polymerase enzymes.
This is particularly important in performing quantitative amplifications where changes in the efficiency of the reaction may well give spurious results. In fact such a nuisance is this that considerable work has gone into discovering enzymes which are less affected by inhibition of blood components. In addition denaturants such as phenol which are very common reagents in DNA extraction are extremely inhibitory to enzyme reactions of all types.
With this in mind methods have been devised to avoid phenol extractions during nucleic acid purification. For example Kogan et al N. Eng. J. Med., 317 16 985-990 (1987) describe extraction of DNA from cells previously separated centrifugally from whole blood by boiling. Others describe similar procedures in EP-A-0237 362 and in Saiki et al Nature, 324, 163-166 (1986). These methods still imply pre-treatment of the whole blood by the rate limiting step of centrifugation and also do not provide the means for the thorough removal of factors found in blood such as haemoglobin which can be very inhibitory to amplification reactions.
Also while seeming to be reasonably quick and easy they are largely useful only for situations where the samples are relatively large. Patent EP 0 393 744 A1 describes yet another version of this which still requires white cell centrifugation and involves adding polysaccharides during the boiling step.
Yet more of a shortcut is the spotting of blood onto filter paper and allowing it to dry. These have been utilised in hospitals for years as "Guthrie" spots and are for the original purposes of screening neonates for disorders such as phenylketonuria, hypothyroidism and galactosemia. A paper reports the extraction of DNA from Guthrie spots by extraction into methanol and subsequent enzyme and phenol/chloroform treatments and also direct amplification by PCR on the membrane after cutting out the spot and fixing with methanol. {McCabe, E. R. B. PCR Methods and Applications 1(2) 99-106 (1991)}. This paper also describes in great detail the need for methods of DNA extraction which lend themselves to non-laboratory collection points without compromising the quality of the result.
This is particularly relevant with neonatal screening programmes, forensic samples, infectious disease situations and agricultural applications in both developed and developing countries world-wide.
For diagnostic testing to be commercially feasible it must also be economically efficient. Each stage must be simple, easy-to-use and consequently safer with respect to possible laboratory infections from occult infectious agents especially in blood, blood products and tissues. The amount of blood or other body fluid contaminated waste must be kept down to a minimum as disposal is difficult and expensive. There is also a need for rapid processing to allow early clinical decisions.
In many clinical samples the sample size is very restricted either due to the moribund state of the patient, the sample origin such as foetuses, amniocentesis samples, cerebrospinal fluid, etc. Also there are many instances where the cells containing the DNA are at low concentration such as in immunosuppressed patients or where the cells are a rare population such as foetal cells in the mothers bloodstream. Further examples of this are in virus infections such as HIV where the virus DNA loading may be extremely small (1 in a million cells for example) but it is still extremely important that this infected cell is found for a correct diagnosis.
Other methods of DNA extraction are employed when it is vital that the DNA remains in relatively large pieces. The largest requirement for synthetic chromosome work requires pieces thousands of megabases long. Other cloning systems need sizes from 50 kb up to 500 kb the larger being the better in many cases as it reduces the total number of clones to cover the entire genome of 3000 megabases. As the entire stretch of DNA within a cell reaches for over 20 metres when released from its protected position within the nucleus it becomes very vulnerable to breakage during pipetting, centrifugation or any other manipulation. Methods have been developed which enclose cell suspensions within agar blocks or sheets and then lysis of the cell cytoplasm and nucleus occurs within the blocks by diffusion of surfactant etc. Other treatments such as enzyme reactions also occur by diffusion within the block so stressing of the DNA is kept to a minmum. Electrophoresis can then be done without any stressful manipulations of the DNA. In this way megabase-sized DNA can be produced for subsequent restriction down to the required size if smaller. McCormick, M. K. et al. P.N.A.S. 86 9991-9995, 1989.
For very delicate, large constructions of DNA such as yeast artificial chromosomes it is crucially important to prevent any breakage during handling. This is done by embedding the DNA within agarose blacks so that it is supported at all times. These agarose plugs can then be placed on an agarose gel and electrophoresis performed as normal for very large DNA samples. [Smith, D. R. et al. P.N.A.S. 87 8242-8246, 1990].
Nuclei isolation is commonly done for a variety of other molecular biology techniques. It has been employed to remove a major source of contamination for mRNA purification. This is an improvement over traditional methods for DNA removal which involve either DNAse treatment which then has to be removed, or guanidinum salts to disrupt the cells followed by physical shearing of the DNA. [Current Protocols in Molecular Biology. Ausubel, F. M. (1988) pp 4.1.2-4.1.6. Wiley, N.Y.]
Other uses for nuclei are, DNA-binding protein studies, in situ hybridisation, transcription studies, nuclear cage studies etc.
Attempts to obtain high molecular weight DNA have been reported. A procedure has been suggested where whole cells are lysed in situ on the membrane and mentions that previously isolated nuclei could also be used though it doesn't say how these nuclei would be prepared, nor is any work reported on this. [Leadon, S. A. and Cerutti, P. A. Anal. Biochem. 120 282-288 (1982)] This paper describes a process where cells are lysed, digested and washed on a polycarbonate filter, allowing contaminating material to be washed off.
The method suggested consisted of the filtration of the nuclei or cells through pores of a much smaller size or simply the drying down of the nuclei onto filter paper. In the first case the amount of nuclei which can be captured is very limited as the pores very quickly block up. Also it is not possible to wash away contaminants very thoroughly due to the nature of the capture. Vigorous washing would remove the nuclei especially if the loading was so great that most of the pores were blocked. Other procedures based on the same principle have a subsequent dialysis stage to selectively remove small cellular contaminants. [De Klowet, D. et al J. Microbial Methods 2. 189-196 (1984)]
These do not involve any specific capture mechanism but rely on non-specific filtration and trapping. The purpose of these methods is to preserve the DNA in an intact form so that any cutting is by design and not due to non-specific breakage during preparation. Similarly there is patent no. JP 2295485 which describes whole blood cell capture by filtration through mesh with pore size smaller than 10 micron. They claim that absorption into the pores of the mesh allows the haemoglobin to be efficiently washed away.
There have appeared in the last few years several new formats for affinity chromatography based on filtration membranes especially for antibody purification. Most commonly these involve the use of a filtration cartridge format, familiar to those working in biological fields. This consists of either a disposable or reusable cassette within which is mounted a disc of filtration membrane.
The membrane is supported on both sides by plastic meshes within the cassette and leading out from the upper and lower surfaces of the cassette is a nozzle designed to be attached to a syringe and an outlet designed to be directed into the collection vessel. These cassettes sometimes include in the design, channels of liquid flow to maximise the interaction of the fluid across the membrane. (U.S. Pat. No. 4,690,757).
Later developments have lead to new versions with either capture moieties already permanently attached or in a chemically activated form for custom derivatisation. The discs are usually about 5 cm in diameter and are claimed to have as high a binding capacity as a column. This is consistent with their use with a syringe for the application of large samples of between 1 and 50 mls of solution at a time.
As these cartridges are contained it is not easy to see when they are full of liquid and this can result in air being drawn through and partial drying out of the membrane in an attempt to reduce the minimum volume. Also because the membranes are contained and supported it is not easy to remove the membrane for visualisation either by light electron microscopy. Similarly they cannot easily be used for subsequent reactions. Some of the cartridges can be disassembled and hence the membrane removed. The true purpose of this is re-use of the cartridge however and usually results in some damage to the membrane.
The concept of effecting separation on the end of a tip has been utilised before in patent No. WO8809201. In this case however the tip contains column material between two frits and is therefore a miniature column.